Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) is the most relevant of the few available experimental animal models of virus-induced demyelination. MS is generally considered to involve an autoimmune pathology, but epidemiological evidence strongly suggests a viral trigger. TMEV are natural mouse pathogens. Intracerebral inoculation of susceptible mouse strains with the BeAn strain of TMEV results in a chronic, progressive, CNS inflammatory demyelinating disorder which is related to life-long persistent CNS virus infection and characterized by spastic hind limb paralysis. TMEV-IDD is considered a highly relevant animal model for multiple sclerosis (MS) since both diseases are characterized by progressive demyelinating lesions with accompanying mononuclear cell infiltrates in which CD4+ T cells and activated microglia/macrophages predominate. Our previous studies have shown that demyelination is initiated by virus specific CD4+ T cells targeting viral epitopes presented in the CNS by microglia/macrophages persistently infected with TMEV. Chronic disease is characterized by the induction of autoimmune responses to a variety of encephalitogenic myelin epitopes which arise via epitope spreading and which appear to play a pathologic role in chronic disease. We will continue to test the overall hypothesis that de novo priming of auto reactive T cells specific for endogenous myelin epitopes occurs both in the CNS and the peripheral lymphoid system and that these autoimmune responses play a major pathologic role in chronic disease progression. Aim 1 will use a variety of approaches to precisely quantitate the temporal appearance of Th1/Th2 cells in peripheral lymphoid organs, peripheral blood and the CNS specific for a panel of encephalitogenic myelin epitopes (using ELISPOT analysis, intracellular cytokine staining, and MHC class II peptide tetramers), determine the T cell receptor (TCR) repertoire (using immunoscope) of these T cells, and assess their encephalitogenic potential (using tolerance and adoptive transfer). Aim 2 will utilize a TCR transgenic T cell transfer system to analyze the temporal appearance and anatomic location of activation of naive myelin-specific T cells stimulated by endogenous myelin epitopes released during acute clinical disease. Aim 3 will determine the relative functional ability of virus-infected peripheral (dendritic cells, macrophages, and B cells) and CNS-resident (astrocytes, microglia, and infiltrating macrophages) APC populations derived from primary cultures and isolated from mice with ongoing TMEV-induced demyelinating disease to produce innate immune cytokines and to process and present TMEV and encephalitogenic myelin epitopes on PLP, MBP, MOG to a panel of myelin epitope-specific naive T cells and activated Th1/Th2 clones. These studies should define the mechanism(s) by which myelin epitope-specific autoimmune responses arise during persistent CNS virus infection and determine their functional contribution to chronic disease. In addition, studies using epitope-specific tolerance to treat chronic TMEV-IDD are applicable for the future design of antigenspecific treatment strategies for MS.